Abstract The long-term goal of the Shlomchik lab is to understand the role of TLRs in SLE. In our previous work, a most surprising finding was that disease was markedly exacerbated in the absence of TLR9, even though TLR9 was required for anti-DNA and anti-chromatin autoantibody production. This has now been confirmed in multiple independent models of murine lupus. The opposite roles for these two similar TLRs is likely a fundamental fea- ture of TLR biology and thus of SLE, yet it is poorly understood. Given the prominent role of TLR signaling in human lupus and the large number of SLE risk alleles that map to the TLR signaling pathway, it is of im- portance to elucidate this fundamental biology and how it applies to disease mechanisms and therapy. The discovery of the key role of TLRs in multiple murine lupus models, coupled with the genetic evidence, has led to the development and testing of TLR inhibitors in the clinic for SLE and other autoimmune diseases, high- lighting the importance. Experiments proposed here will directly address how TLR7 and TLR9 have divergent roles, how they cross-regulated each other, and how this promotes and regulates disease. Our lab has pio- neered genetic in vivo methods for the study of lupus mechanism in animal models, in part leading to the recognition of the roles of TLR7 and TLR9, in studies supported in the prior period of this PPG. We have creat- ed novel models and used genetic approaches in order to dissect the cells and molecular signaling pathways required to promote disease in vivo. Here we continue to make novel models and study them in vivo, coupled with studies of the basic signaling and biology of TLR7 and 9 at the cellular and biochemical levels. We hypothesize that differential tissue-specific expression and signaling can explain the disparate global ef- fects of TLR7 and TLR9 deletion, an idea supported by our preliminary data with new TLR9 ?floxed? mice. To address this, we will manipulate the expression of TLRs in vivo in key cell types. In Aim 1 we will test the hy- pothesis that tissue-specific expression of TLR7 and TLR9 explains some or all of their divergent phenotypes. We will extend our work on tissue-specific TLR9 expression to both macrophages and neutrophils and to addi- tional lupus models. We will make new floxed and rosa26 conditional mice to allow TLR7 tissue-specific ex- pression. This will let us study the yin-yang of TLR7 and TLR9; to date we have only looked at TLR9. In Aim 2 we will address the hypothesis that TLR9 regulates TLR7 signals in a cell intrinsic way. To test this, we have made several tools, including mutants of TLR9 and TLR7, and will make additional mutants and mutant mice. Our unique focus will be on primary B cells, enabled by Aim 1. This will allow us to determine both in vivo and in vitro if TLR9 suppresses TLR7 signaling by competing for trafficking molecules, for signaling molecules or by inducing the expression of counter-regulatory proteins. In addition, we will test whether the cytoplasmic tails of TLR7 and TLR9 mediate different signaling. Finally, in Aim 3, we will test the therapeutic potential of TLR inhi- bition using an inducible Cre to delete MyD88 and thus end TLR7/9 signaling at different stages of disease.